PROJECT SUMMARY The oncogenic receptor, HER2, is overexpressed in 25-30% of breast cancer patients and is characterized by aggressive growth, increased disease recurrence, and poor prognosis. While much of the signaling mechanism(s) have been elucidated, the impact of HER2 positivity on cellular metabolism is not well understood. Recently, we found high intracellular accumulation of lactate that correlated with HER2 positivity in breast cancer cells. Lactate accumulation within tumor tissue is well known (Warburg effect) and largely due to the increased glycolytic rate of cancer cells. The major lactate dehydrogenase enzymes (LDHA and LDHB) are responsible for conversion of pyruvate to lactate and often overexpressed in cancer. This evidence points to the alarming clinical problem: is HER2 positivity a driver towards derailed metabolism and a potentially more proliferative/aggressive phenotype? We hypothesize that HER2 expression is the driver for a glycolytic phenotype in a subset of breast cancer that leads to lactate accumulation. We propose three interlinked aims to systematically define the biochemical impact of HER2 overexpression to drive breast cancer cell survival through identifying and tracking key metabolites in the glycolytic pathway of lactate, and understand how anti- HER2 strategies promote cell death in specific experimental cellular contexts: 1) determine the metabolic path of lactate in HER2-positive breast cancer cells, 2) determine the metabolic impact of anti-HER2 strategies, and 3) define target candidate expression in altered metabolic pathways. A combination of 3D/spheroid breast cancer models with variable and inducible HER2 expression will be used with metabolic tracers (13C)-glucose, - lactate, -glutamine to define consumption and release profiles over time by unbiased, data-driven NMR spectroscopy. This proposal is significant because it seeks to reveal the molecular mechanism between HER2 and lactate using a metabolomic approach in a robust isogenic 3D culture. Outcomes may directly impact the selection of protein and small molecule pharmacologic tools to better understand lactate shuttling in human malignancies. Furthermore, this is responsive to the limited scope, URM student studies targeted for the SC3 initiative and it is imperative for the critical path to show proof-of-concept with in vitro-based spheroid studies before translating into advanced models such as human breast cancer samples. .